Microbiology 101 – The Gram Stain

The Gram stain: even many lay people have heard of this basic but incredibly important test that serves an integral role in the microbiology laboratory. Here I would like to give a brief overview over the basics of the Gram stain.

The staining procedure for bacteria was discovered over 100 years ago by Hans Christian Gram. Almost all clinically important bacteria can be detected using this stain. Exceptions are few and include intracellular bacteria like Chlamydia, bacteria that lack a cell wall such as mycoplasma and spirochetes like Treponema which due to their dimensions cannot be resolved by a light microscope.

The Gram stain divide bacteria into two major categories; gram positive, which takes up and retains the primary stain, and gram negative, which the primary stain can be washed out by a decolorizing acetone-alcohol.

How do you perform the procedure?

First you must fix the specimen to be examined (from a clinical source or from an agar plate) to the microscope slide either by heating the slide or by the use of methanol.

After the specimen is fixed, flood the slide with the primary stain, crystal violet (purple). Here the bacterial cells are filled with stain. Rinse the slide with water.

Next flood the slide with Gram’s iodine. A crystal violet/iodine complex is formed in the cell. Rinse the slide.

Wash the slide with an organic solvent (acetone-alcohol) decolorizer and finally stain with a counterstain, safranin (red).

How come some bacteria stain gram positive and others gram negative?

This is due to different cell wall composition with the two bacterial groups. A gram positive organism, e.g. Staphylococcus aureus will retain the crystal violet because of a thick peptidoglycan layer and teichoic acid cross-links (cells appear purple microscopically).

Gram negative bacteria, e.g. E.coli lack this cell wall and the crystal violet/iodine complex is leeched out of the bacterial cell during decolorization. The acetone-alcohol disrupts the outer membrane of the gram negative cell causing the decolorization. After decolorization of gram negative bacteria, the cells become colorless and are subsequently filled with safranin (red) giving it the gram negative appearance microscopically (pink-red).

A Gram stained smear is examined using the oil immersion objective (1000x). Not only do you report the Gram reaction (positive or negative) but also the bacterial morphology (cocci or bacilli) and any formations (clusters or chains for example).

How is the Gram stain useful with clinical samples?

Often times treatment decisions can be made based on the Gram stain result alone. One example is in a critical culture site like blood or CSF where time is of the essence, a gram stain of gram positive cocci in clusters can let the treating physician know the he/she is dealing with a staphylococcus species and an appropriate antibiotic for staph can be started while waiting for the culture result.

The Gram stain is one of the most important laboratory procedures in the clinical microbiology laboratory and may appear to be deceptively simple. Instead, a technologist who is very proficient at reading Gram stained smears typically has considerable experience and training.